Drug development is an advanced country-type strategic process requiring a massive commitment of time and money of more than ten years and eight hundred million dollars, respectively. A well-developed social infrastructure is also necessary for drug development.
Broadly, the process of drug development can be divided into the discovery of a drug target by basic research, the selection of effective and lead materials by compound screening, the determination of a candidate drug, clinical research through pre-clinical work/clinical phase 1, and commercialization through clinical phases 2 and 3.
Of a total of 35,000 genes discovered thus far as drug targets, approximately 500 are currently under research for drug development, with a steady expansion of the development subject since the Human Genome Project. Once a drug target is selected, development of a screening method that is the most suitable and effective should be undertaken. The screening method can be divided into an in vitro assay and a cell-based assay. Major pharmaceutical companies possess libraries of compounds, typically amounting in number to ten of thousands to hundreds of millions, as screening targets, and such a number of compounds are employed from an early screening stage.
A great expense for this screening process has given rise to making every effort to design effective screening methods and develop high-speed and minimized apparatuses and reagents which allow the screening of as many compounds as possible within a short period of time.
A screening process for many compounds must be technically simple with high reproducibility. When a drug target is an enzyme, a relative easy approach is possible thanks to an abundant number of screening methods and reagents established therefor. However, because most of the biological processes taking place within cells are associated with interaction with proteins, a screening method based on interaction with proteins is the most effective among analysis methods for developing lead compounds. Great weight is given to such screening methods for the following reasons: a protein functions as it associates with another protein in vivo; a change in gene and protein expression, in intracellular location, and/or in structure through post-translational modification induces an altered interaction between proteins, resulting in a change in the activity and regulation of intracellular metabolisms and signaling pathways; and an abnormal protein interaction attributed to a genetic mutation directly leads to the onset of a disease. There are technologies for detecting protein interactions, including FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer) and FP (Fluorescence Polarization), and a technical advance has also been achieved in apparatuses to which the technologies are applicable. In recent years, HCS (high-content screening) with automated high resolution microscopy has been introduced, whereby after cells are incubated with substances in multiwell plates, such as 96-well, 384-well plates, etc., phenomena associated with the quantitative change and transport of proteins within cells can be rapidly observed in a quantitative manner. HCS is now arising as the most interesting biological research method for world-leading pharmaceutical companies or research institutes because it allows the quantitative analysis of biological parameters, such as protein interaction, Ca++ influx, etc., which are difficult to screen on a large scale with conventional methods, over the simple information obtained using conventional enzyme detection methods or reporter systems, for example, on enzyme activity, promoter strength, protein levels, etc.
Typically, a procedure for drug screening comprises preparation of compound aliquots, dilution, mixing of screening components, culturing and detection, analysis of screening data, and reporting on results. A high-throughput screening (hereinafter referred to as “HTS”) system is used to rapidly process such a serial procedure. Advanced pharmaceutical companies are known to possess a compound library consisting of hundreds of millions of different compounds, and whenever a novel drug target is discovered, the companies take advantage of the HTS system in screening the compound library against the drug target. Thus, major pharmaceutical companies have accumulated tremendous data on biological activities of hundreds of millions of compounds, thus far. In order to more rapidly and effectively screen the compound library against thousands of drug targets, a curve-fitting tool capable of performing various functions including a QC function, error checking for overlapped data, calculation of relative activity (% activity), and extraction of biochemical parameters, such as IC50, Ki, and Km, is needed. In this regard, HTS which allows much data to be produced by one screening process is required. This new technology, aiming to overcoming problems associated with the conventional technology, is basically designed to evaluate synthetic compounds randomly on a mass scale through automation, and can reduce the time taken to determine candidate drugs as much as possible in association with automated synthesis of new materials (CCL), molecular design and systematic information management.
Prerequisites for HTS with a capacity of screening more than 10,000 different compounds a day are summarized as follows:
(1) Rapidity: Given a higher screening speed, an HTS can screen a higher number of compounds, and thus can complete its performance within a shorter time and at a lower expense.
(2) Expense: Reagents used in the screening process account for a large portion of the total screening expense. A measure must be taken toward financial retrenchment.
(3) Miniaturization: Miniaturization is not only one of the best measures to cut expenses for reagents, but can also reduce the time taken to perform a screening process. Besides, it can reduce laboratory space necessary for the instruments.
(4) Automation: Automation increases reproducibility of results as well as the speed of screening. Particularly, it makes a great contribution to the reduction of experimental error.
(5) Screening sensitivity: The sensitivity of a detection method is directly relevant to the quantity of samples to be used. High detection sensitivity is required because it takes a longer time to screen samples of lower sensitivity.
(6) Non-radioactive method: As high as 50% of the HTS methodologies developed thus far use radioactive substances. However, radioactive substances produce waste which must be specifically cared for, and thus are disadvantageous in terms of space, time and finances.
(7) Simplicity: Because a method operating with filtration, separation, washing, distinction, and solid-state extraction requires additional expense and processes, the screening process should be simplified in a liquid state as much as possible.
Pharmaceutical companies have made enormous investments in the development of chemical approaches to compounds, and HTS technology. As a result, the number of drug candidates has sharply increased. Then, the candidates excavated through the primary screening process (discovery and evaluation of target, and excavation of candidates) are subjected to a secondary screening process (optimization of candidates) which is much lower in yield than is the primary screening process. The difference of yield between the primary and secondary screening processes incurs a significant bottleneck phenomenon in the development of new drugs. Hence, it is an important challenge throughout new drug development to increase the efficiency of secondary screening to a level in harmony with the primary screening process without deteriorating the quality of data generated in the secondary screening.
High-content screening (HCS) can be defined as a “technology for functionally and complexly screening various targets inside living cells on the basis of highly temporally and spatially resolved fluorescence images.” Among fundamental technologies of HCS are a cell-based assay, real-time fluorescent imaging of living cells with high temporal and spatial resolution, and a high-speed and high-content automated assay. Representative of HCS analysis instruments is the Opera system of Perkin-Elmer shown in FIG. 1. Formal cell analysis data obtained by the Opera system is as shown in FIG. 2. In this regard, first, images of tens of aggregated cells are obtained within a field, and cell nuclei and walls are discriminated among the images, during which images of some cells are removed on the program while leaving significant cell images. Finally, two-color images are obtained as seen in FIG. 2.
The high-content screening technology has been based on fluorescence assay, so far. However, fluorescent labels used in fluorescence assay weaken in fluorescence intensity (photobleaching), and exhibit interference between different fluorescent labels because excitation light with a very narrow wavelength range is used while the fluorescent light has a very broad range of wavelengths. In addition, there are an extremely limited number of available fluorescent substances.
Therefore, there is a need for a new method for effective high-speed drug screening that exhibits sharp spectrum peaks without causing interference between fluorescent substances, thus allowing the detection of multiple drugs.
In recent years, Raman spectroscopy has attracted extensive attention.
Inter alia, Surface Enhanced Raman Scattering (SERS) is a spectroscopic method which utilizes the phenomenon whereby, when molecules are adsorbed on a roughened surface of a metal nanostructure such as a gold or silver nanoparticle, the intensity of Raman scattering is dramatically increased to the level of 106˜108 times compared with normal Raman signals. As light passes through a transparent medium, molecules or atoms of the medium scatter the light. In this regard, a small fraction of the photons undergoes inelastic scattering, known as Raman scattering. For example, a fraction of the incident photons interact with the molecules in such a way that energy is gained or electrons are excited into higher energy levels, so that the scattered photons have a different frequency from that of the incident photons. Because the frequencies of the Raman scattering spectrum account for the chemical compositions and structural properties of the light absorbing molecules in a sample, Raman spectroscopy, together with the nanotechnology which is currently being quickly developed, can be further developed for highly sensitive detection of a single molecule. In addition, there is a strong expectation that an SERS sensor can be importantly used as a medical sensor. The SERS effect is in relation with plasmon resonance. In this context, metal nanoparticles exhibit apparent optical resonance in response to incident electromagnetic radiation due to the collective coupling of conduction electrons within the metal. Thus, nanoparticles of gold, silver, copper and other specific metals can fundamentally serve as nanoscale antenna for amplifying the localization of electromagnetic radiation. Molecules localized in the vicinity of these particles show far greater sensitivity to Raman spectroscopy.
Accordingly, many studies are being actively carried out about using SERS sensors to detect biomarkers including genes and proteins for early diagnosis of various diseases. Raman spectroscopy has various advantages over other methods (e.g., infrared spectroscopy). While infrared spectroscopy can detect strong signals from molecules which have a dipole moment, Raman spectroscopy allows strong signals to be detected even from non-polar molecules in which induced polarizability is modulated. Hence, almost all organic molecules have their own Raman shifts (cm−1). In addition, being free from the interference of water molecules, Raman spectroscopy is suitable for use in the detection of biomolecules including proteins, genes, etc. Due to low signal intensity, however, the stage of development of Raman spectroscopy has not yet reached the level where it can be used in practice in spite of research spanning a long period of time.
Since its discovery, Surface-Enhanced Raman Scattering (SERS) has continually been developed to such a level so as to detect signals at a molecular level from randomized aggregates of fluorescent dye-absorbed nanoparticles (Science 1997, 275(5303), 1102; Phys rev lett 1997, 78(9), 1667). Since then, many studies of SERS enhancement with various nanostructures (nanoparticles, nanoshells, nanowires) have been reported. In order to utilize SERS as a highly sensitive detection method for a biosensor, Mirkin et al. reported highly sensitive DNA analysis by using DNA-modified gold nanoparticles, with a detection limit of 20 fM (2002, Science, 297, 1536). However, there have been almost no advances in preparing single molecule SERS active substrates based on the salt-induced aggregation of silver (Ag) nanoparticles having Raman active molecules (e.g., Rhodamine 6G) since the first study. A report has it that only a fraction (less than 1%) of heterogeneously aggregated colloids has single molecule SERS activity (J Phys Chem B 2002, 106(2), 311). Like this, randomly roughened surfaces provide a multitude of interesting essential data associated with SERS, but this strategy is fundamentally impossible to reproduce because even a small change in surface morphology leads to a significant change of enhancement. Recently, Fang et al. reported a quantitative measurement of the distribution of site enhancements in SERS. The hottest SERS-active sites (EF>109) accounted for only 63 sites out of a total of 1,000,000 sites, but contributed 24% to the overall SERS intensity (Science, 2008, 321, 388). In these regards, assembling SERS-active nanoparticles into well-defined and reproducible hot SERS nanostructures would lead to a highly reliable, sensitive assay for biomolecules and be greatly useful for use in xenodiagnosis and in vivo imaging techniques.
Leading to the present invention, intensive and thorough research into the high-speed screening of multiple drugs in association with Raman spectroscopy, conducted by the present inventors, resulted in the finding that when exposed to a sample containing one or more analytes, a nanoparticle labeled with an analyte-recognizing biomolecule functionalized thereon, comprising a core and a shell with a nanogap formed therebetween, is used to produce Raman signals if it is irradiated with an excitation laser beam, and that specific Raman wavelengths can be obtained from the Raman signals by filtration through multiple Raman filters, detected with a high SERS enhancement factor by a detector, and color coded to generate color-coded Raman images, whereby multiple drugs can be screened at high speeds with high reproducibility and reliable quantifiability.